This research focusses on oxidative modification of biopolymers, especially of proteins. The reaction is typically initiated by the binding of a metal such as iron or copper to a cation binding site on the targeted protein. Oxygen reacts at that site to generate an activated species which then oxidizes amino acid residues at the binding site. This covalent modification has been implicated in important physiologic and pathologic processes. These include the aging processes, atherosclerosis arthritis, carcinogenesis, gene regulation, hypertension, intracellular protein turnover, oxygen toxicity, and reperfusion injury after ischemia. Determination of the actual roles of oxidative modification in these processes requires the application of specific assays for modified proteins, identification of the structural and functional changes induced by modification, and understanding of factors which modulate the rate and specificity of oxidative modification in vivo. These are the current aims of the project. We continued development of methodologies for assessing oxidative modifications, including an automated HPLC technique capable of analyzing complex samples. This method was applied to a model of neonatal cerebral ischemia and demonstrated that reflow causes substantial oxidative modification of proteins in the ischemic hemisphere. Notably, the contralateral, control hemisphere suffered almost equal oxidative damage. Two circulating proteins were shown to be sensitive to oxidative modification and to lose function as a consequence. Fibrinogen was modified by two different systems, each of which led to loss of clot formation upon exposure to thrombin. Thrombin still cleaved fibrinopeptides normally, leading to the conclusion that the modified fibrin was unable to aggregate into a clot. Thus, oxidative modification of fibrinogen produces a dysfibrinogen. Alpha-2-macroglobulin is a major circulating antiprotease which loses function upon exposure to activated neutrophils or a model system simulating the neutrophils. Inactivation was associated with loss of a single tryptophan residue, and spectrophotometric analysis suggests conversion of tryptophan to N- formyl-kynurenine. Glutamine synthetase continues to be studied as a model system for metal- catalyzed oxidative modification of proteins. Mass spectral analysis of peptides from the modified protein demonstrated the conversion of a specific histidine residue to 2-oxo-histidine, an unusual amino acid which could therefore serve as a marker for oxidative damage or oxidative stress. The oxo-histidine residue was also identified by Edman sequencing of the modified protein.